CRISPR Cas genes and Legionella pneumophila infection

CRISPR Cas基因与嗜肺军团菌感染

• 批准号: 8424733
• 负责人: NICHOLAS P CIANCIOTTO
• 金额: $ 21.78万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-12 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8424733
• 关键词: Acanthamoeba castellanii; Aerosols; Alveolar Macrophages; Amoeba genus; Animal Model; Archaea; Area; Bacteria; Bacteriophages; Biochemical; Biological Assay; Biology; Breathing; Communities; Complement; Data; Defect; Development; Devices; Disease; Eubacterium; Fostering; Genes; Genomics; Growth; Habitats; Human; Immunity; Infection; Lead; Legionella pneumophila; Legionnaires' Disease; Link; Lung; Microbial Genetics; Mutation; Nature; Organism; Parasites; Pathogenesis; Pathogenicity; Plasmids; Play; Pneumonia; Process; Proteins; Protozoa; RNA; Research; Research Project Grants; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Risk; Role; Sequence Analysis; Staging; System; United States National Institutes of Health; Virulence; Virulent; Water; Work; design; extracellular; human BCAR1 protein; innovation; intracellular parasitism; killings; macrophage; mutant; novel; pathogen; pathogenic bacteria; programs; public health relevance; transmission process

DESCRIPTION (provided by applicant): Arguably one of the major new findings in microbial genetics in recent years is the discovery of the CRISPRCas system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci are novel sequences found in Archaea and Bacteria that are comprised of palindromic repeats separated by unique spacer sequences. Upstream of the CRISPR array are the CRISPR-associated sequences (cas) genes. CRISPRCas loci have been primarily implicated in immunity to phage and plasmid. Overall, CRISPRCas loci have been studied in only a relatively small number of model organisms. However, genomic sequencing indicates that CRISPRCas loci are found in >400 different bacteria, including in a variety of pathogenic organisms and intracellular parasites, and the potential role of CRISPRCas in pathogenicity and intracellular infection has not been directly examined. Legionella pneumophila (Lpn) is the bacterial agent of a severe form of pneumonia called Legionnaires' disease. Humans come in contact with the bacterium by inhaling contaminated water droplets. In its aquatic habitats, Lpn is an intracellular parasite of amoebae, and infection of these protozoa is unquestionably an important step in the transmission and pathogenesis of disease. In the lung, Lpn replicates in macrophages in a process that mimics infection of amoebae. Virulent Lpn strains contain a type of CRISPRCas locus that has not been previously investigated. Given this as well as the pathogenic and intracellular nature of Lpn, we embarked on an examination of the role of CRISPRCas in Lpn biology and virulence. Data presented here show that the CRISPRCas locus of Lpn strain 130b is expressed under extracellular and intracellular growth conditions. Furthermore, loss of cas2 results in a dramatic (approx. 1000-fold) defect in Lpn infection of the amoeba Acanthamoeba castellanii. These observations lead to the unique hypothesis that the Cas protein(s) of Lpn play a heretofore unrecognized role in infection that is outside of a role in immunity to phage and plasmid. Thus, Aim 1 will utilize a full panel of CRISPRCas mutants and complemented mutants in infection assays to determine the importance of the entire CRISPRCas locus in intracellular infection. Given the now established importance of Cas2 in Lpn infection, Aim 2 will use a biochemical approach to elucidate the function of Cas2 by determining if the protein has RNase activity and if this activity is important for infection. Lastly, Aim 3 will identify the stage(s) of infection n which Cas2 plays a role; e.g., the cas2 mutant's infection defect could be indicative of diminished resistance to intracellular killing and/or impaired intracellular replication. Thus, by using Lpn a a new model organism, we will probe for novel functions of the CRISPR/Cas locus that seek to shift current research paradigms.

描述（由申请人提供）：可以说，近年来微生物遗传学领域的主要新发现之一是 CRISPRCas 系统的发现。 CRISPR（成簇规则间隔短回文重复序列）基因座是在古细菌和细菌中发现的新序列，由由独特间隔序列分隔的回文重复序列组成。 CRISPR 阵列的上游是 CRISPR 相关序列 (cas) 基因。 CRISPRCas位点主要涉及对噬菌体和质粒的免疫。总体而言，仅在相对少数的模式生物中研究了 CRISPRCas 位点。然而，基因组测序表明，CRISPRCas位点存在于超过400种不同的细菌中，包括多种病原生物和细胞内寄生虫，并且CRISPRCas在致病性和细胞内感染中的潜在作用尚未得到直接检验。嗜肺军团菌 (Lpn) 是一种称为军团病的严重肺炎的细菌病原体。人类通过吸入受污染的水滴来接触细菌。在其水生栖息地中，Lpn 是变形虫的细胞内寄生虫，这些原生动物的感染无疑是疾病传播和发病机制中的重要一步。在肺部，Lpn 在巨噬细胞中复制，其过程类似于阿米巴原虫感染。强毒 Lpn 菌株含有一种以前未曾研究过的 CRISPRCas 基因座。鉴于这一点以及 Lpn 的致病性和细胞内性质，我们开始研究 CRISPRCas 在 Lpn 生物学和毒力中的作用。这里提供的数据显示 Lpn 菌株 130b 的 CRISPRCas 基因座在细胞外和细胞内生长条件下表达。此外，cas2 的缺失会导致阿米巴棘阿米巴 (Acanthamoebacastellanii) 的 Lpn 感染出现显着的（约 1000 倍）缺陷。这些观察结果得出了一个独特的假设，即 Lpn 的 Cas 蛋白在感染中发挥着迄今为止未被认识的作用，该作用超出了对噬菌体和质粒的免疫作用。因此，目标 1 将在感染测定中利用全套 CRISPRCas 突变体和互补突变体，以确定整个 CRISPRCas 位点在细胞内感染中的重要性。鉴于现已确定 Cas2 在 Lpn 感染中的重要性，Aim 2 将使用生化方法通过确定该蛋白质是否具有 RNase 活性以及该活性是否对感染很重要来阐明 Cas2 的功能。最后，目标 3 将确定 Cas2 发挥作用的感染阶段；例如，cas2突变体的感染缺陷可能表明对细胞内杀伤的抵抗力减弱和/或细胞内复制受损。因此，通过使用 Lpn 作为一种新的模式生物，我们将探索 CRISPR/Cas 基因座的新功能，以寻求改变当前的研究范式。